We have developed novel approaches to create and evaluate murine immunodeficiency models and to identify genes potentially involved in such diseases. In particular, we have developed the RAG-2 deficient blastocyst complementation method to rapidly assess the role of almost any gene in lymphocyte differentiation of function. We will use these methods to generate models for certain human immunodeficiencies that affect lymphocyte differentiation and/or function. The human btk gene is implicated in X-linked agammaglobulinemia (XLA). We will determine the structure of the murine btk gene and then use gene targeted mutation techniques to generate mice that lack a functional btk gene in their lymphocytes (via RAG-2 deficient blastocyst complementation) or in their germline. These mice will serve as a murine model for XLA and as an experimental system to study the function of the btk protein. To elucidate the function of the btk protein, mutated forms of the btk gene will be used to replace the inactivated btk gene either by transfection of appropriated constructs into mutant ES cells and RAG-2 deficient blastocyst complementation or by transgenic complementation of btk germline mutations. Mice carrying the mutated forms of btk will be assayed for phenotype characteristics and used as a source of cells for biochemical studies of btk function. We also have employed a simple assay to rapidly identify mutations that impact on the VDJ recombination reaction. As a means to identify genes potentially involved in human SCID diseases, we will employ mutant CHO cells to identify, isolate, and characterize genes involved in VDJ recombination and DNA repair, such genes have been implicated in the mouse scid defect. We have already defined 3 such genes based on ability to complement CHO DNA repair/VDJ recombination defects and demonstrated that two of these map to human chromosomes 2 and 5, respectively. Once isolated, these genes will be disrupted in ES cells and assayed for affects on lymphocyte development by RAG-2 deficient blastocyst complementation. We will similarly assay cells from human severe combined immunodeficient patient (that are not X-linked of ADA-deficient) for defects in VDJ recombination with the ultimate goal of elucidating the involved gene.